Cell analysis device, apparatus, and cell analysis method using same

ABSTRACT

The purpose of the present invention is to provide a single cell analysis device in which the improvement of the nucleic acid capturing efficiency and the improvement of the cell capturing efficiency are both achieved and a highly accurate single cell analysis data is thereby obtained. The present invention relates to an improvement of a cell analysis device including a two-dimensional array chip having a plurality of cell capture parts capable of capturing a single cell in each of the capture parts, and nucleic acid capture parts corresponding to the respective cell capture parts, the nucleic acid capture parts being capable of capturing a nucleic acid extracted from the cell captured by the cell capture part.

TECHNICAL FIELD

The present invention relates to the technical fields of gene expressionanalysis, cell function analysis, biological tissue analysis method,disease diagnosis, drug discovery, and the like, and more specifically,relates to a cell analysis device, an apparatus and a cell analysismethod using the same which makes the genetic analysis for a single cellpossible.

BACKGROUND ART

Single cell analysis is a technique for detecting and/or quantifyingwith high accuracy biomolecules in cells for every single cell. Toperform the single cell analysis, it is necessary to isolate cells forseparate treatments, efficiently extract nucleic acids to be measuredfrom the cells, synthesize a complementary chain (for example, cDNA),and if needed, perform sequence analysis of a product obtained byamplification.

Patent Document 1 discloses a device configured to capture respectivecells in each pores of a porous array sheet, subsequently, capture anucleic acid derived from the cell with a DNA probe that is immobilizedin the pore and that has a different tag sequence for each pore tosynthesize cDNA, and thus can obtain a product for sequence analysiscapable of distinguishing from which cell the captured nucleic acid hasbeen derived. The basic configuration (corresponding to FIG. 8 of PatentDocument 1) of such device is shown in FIG. 1.

In the device of FIG. 1, a cell solution containing cells 101 isintroduced from an inlet 106. The cell solution is sucked from an upperoutlet 107 in order to fill an upper region 104 of a porous membrane 102having a planar substrate shape with the cell solution. If a negativepressure is applied by sucking the solution from a lower outlet 108, thecell solution is sucked through the porous membrane 102, and the cells101 are guided to a cell capture part 103. The cells 101 are captured bya lattice-shaped cell capture part 103 constructed on the porousmembrane 102, and then, by disrupting the cells, the nucleic acids (forexample, mRNA) within the cells are captured by a DNA probe (forexample, a poly-T probe) immobilized within the porous membrane directlybeneath the cells.

By using a device as shown in FIG. 1, the nucleic acids extracted fromthe captured cells can be captured with hardly any contact with regionsother than the inner wall of the porous membrane which is the reactionregion, and the nucleic acid (for example, cDNA) corresponding to thecaptured nucleic acid can be synthesized. Therefore, the probabilitythat nucleic acids are adsorbed on the inner wall of the deviceunrelated to the reaction can be reduced, and a product for sequenceanalysis can be prepared highly efficiently.

PRIOR ART DOCUMENT Patent Document

-   Patent Document 1: WO2014/020657 A

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

To efficiently capture nucleic acids in the device as shown in FIG. 1,it is necessary that the average pore diameter of the pores constitutingthe porous membrane 102 is several μm or less, and especially, 1 μm orless is preferable. Further, it is desirable that the thickness of theporous membrane 102 is 10 μm or more, especially, several tens of μm ormore. However, if such porous membrane is used, the pressure loss whenthe cell solution passes therethrough becomes large. If the pressureloss becomes large, the suction rate decreases upon suctioning thesolution from the lower outlet 108. This results in that the suctionrate of suctioning the cells 101 into the cell capture part 103decreases. Therefore, the phenomenon that a relatively large number ofcells settle with gravity, and remain in the other regions on the devicebefore reaching the cell capture part 103 has been observed. The cellindicated by 109 in FIG. 1 is one example of a cell which did not reachsuch cell capture part 103.

The cells remaining in the regions other than the cell capture part 103not only reduce the ratio of cells that can be analyzed but also causean additional problem. That is, when extracting nucleic acids fromcells, a step for disrupting the cells is necessary. However, upon that,cells present in regions other than the cell capture part 103 are alsodisrupted. Owing to this, there arises a problem that the nucleic acidsextracted from the cells flow in a plurality of cell capture parts 103and it becomes difficult to perform an accurate single cell analysis.

The above problems are caused by an increase of the pressure loss due tothe increase of the membrane thickness and a decrease of the porediameter of the porous membrane 102, which are intended for improvingthe nucleic acid capturing efficiency. Therefore, the problemessentially lies in the fact that the improvement of the nucleic acidcapturing efficiency and the improvement of the cell capturingefficiency are in a trade-off relationship.

Furthermore, a nucleic acid amplification step is necessary to performsequence analysis after the capturing of the nucleic acid. In this step,it is necessary that the nucleic acid amplification product is releasedfrom the nucleic acid capture part (as described in Patent Document 1),extracted to the outside of the device as the solution sample, andsequence analysis is performed. There may arise a problem that theamplification product within this solution is adsorbed on the inner wallof the device. The yield of the nucleic acid amplification productnecessary for sequence analysis is reduced by adsorption. In addition,since the adsorption rates differ in accordance with the difference inthe base length of the nucleic acids, sequence analysis may possibly beperformed with a composition which is different from the composition ofthe original nucleic acid in the cell.

Means for Solving the Problem

The present inventors examined the above-mentioned problems, and as aresult, found the followings. It is effective to make this devicestructure such that a repulsive force which causes the cells not toapproach the region on the substrate other than the cell capture part isapplied to the cells when a cell solution is sucked from the backsurface of the substrate (for example, the porous membrane) on which thenucleic acid capture part is provided. Namely, as one example, thedevice configured so as to apply gravity to the cells in a directionopposite to the suction direction to the cells as shown in FIG. 2 iseffective. In addition to the gravity as exemplified in FIG. 2, anelectrostatic force caused by the surface treatment or anelectrophoretic force can be effectively adopted as such repulsiveforce.

Further, the present inventors found that the method of using a secondthree-dimensional porous membrane (three-dimensional porous member) asshown in FIG. 3 to strengthen the suction force to thereby preventadsorption in the regions other than the cell capture part due to otherforces. However, since the inner wall of the second three-dimensionalporous membrane needs to be hydrophilic, it is highly likely that a DNAstand having a negative charge will be adsorbed on the inner wall.Therefore, the yield of the obtained amplification product may decrease,and a change in the yield due to the length of the DNA amplificationproduct may occur, thus, the analysis of the nucleic acid composition ina single cell may be difficult. To further improve this point, it hasbeen found that it is effective to provide a means for separating thehydrophilic three-dimensional porous membrane and the solution of thenucleic acid capture part which becomes the amplification reaction partin the amplification reaction process. As the means for performing suchseparation, it has been found that it is effective to provide a meansfor injecting air or a nonpolar solvent such as oil at an appropriatetime between a first porous membrane (or substrate, i.e., thetwo-dimensional array chip) which is the nucleic acid capture part and asecond hydrophilic three-dimensional porous membrane for suctionassistance, or, provide an actuator inside the device that causes thedistance between the placed first and the second porous membranes in avertical direction with respect to the membranes becomes large.Furthermore, it has been found that it is effective to use a separationmembrane such as an ultrafiltration membrane or gel membrane between thefirst and the second porous membranes such that the amplificationproduct does not reach the second porous membrane.

Therefore, the summary of the present invention is as follows:

(1) A cell analysis device comprising:

a solution introduction channel for introducing a solution containingcells;

a substrate having a plurality of sets of a cell capture part and anucleic acid capture part, the cell capture part being in contact withthe solution introduction channel and having a concavity capable ofcapturing a single cell, and the nucleic acid capture part beingprovided in a corresponding manner to the concavity of the cell capturepart and capturing a nucleic acid extracted from the cell having beencaptured by the cell capture part;

a discharge channel that is provided adjacent to the nucleic acidcapture part of the substrate, and discharges a solution of the nucleicacid capture part; and

a pressure control means provided in the discharge channel,

in which the substrate has a repulsive force in a direction whichseparates the cells from the substrate, and

the pressure control means controls such that, when the cell is capturedby the cell capture part, a force in the direction from the cell capturepart to the nucleic acid capture part serves as a first pressure that islarger than the repulsive force.

(2) The cell analysis device according to (1), in which the pressurecontrol means controls such that, when performing the nucleic acidreaction in the nucleic acid capture part, the force in the directionfrom the cell capture part to the nucleic acid capture part is largerthan gravity and smaller than the first pressure.

(3) The cell analysis device according to (1), in which a region whichis on the substrate other than the concavity and which is in contactwith the solution containing the cells is a region that exerts arepulsive force on the cells.

(4) The cell analysis device according to (1), in which the repulsiveforce is a repulsive force caused by the substrate being placed in sucha manner that the cell capture part captures the cell in the directioncounter to gravity.

(5) The cell analysis device according to (1), in which the repulsiveforce is a repulsive force caused by the surface of the substrate havingbeen subjected to a treatment which prevents the adsorption of cells.

(5-1) The cell analysis device according to (5), in which the treatmentwhich prevents the adsorption of cells is a surface treatment by acoating agent, for example, MPC polymer.

(6) The cell analysis device according to (1), further comprising anelectrode pair provided so as to sandwich the substrate, in which thenucleic acid capture part comprises metal microparticles, and therepulsive force is a repulsive force caused by a voltage applied to theelectrode pair and dielectric coupling by the metal microparticles.

(6-1) The cell analysis device according to (6), in which the metalmicroparticles are gold microparticles, and/or the substrate is aplatinum substrate.

(7) A cell analysis device comprising:

a solution introduction channel for introducing a solution containingcells,

a two-dimensional array chip having a plurality of cell capture partsand nucleic acid capture parts, the cell capture parts being providedadjacent to the solution introduction channel and each being capable ofcapturing a single cell, and each of the nucleic acid capture partsbeing provided corresponding to each cell capture part of the pluralityof cell capture parts and capturing a nucleic acid extracted from thecell having been captured by the cell capture part,

a discharge channel having a three-dimensional porous body which absorbsa solution retained in the nucleic acid capture part and for dischargingthe solution, and

a separation control part for controlling separation between the nucleicacid capture part and the discharge channel,

in which the separation control part performs separation control suchthat, after the nucleic acid is captured by the nucleic acid capturepart, a product amplified from the captured nucleic acid is notintroduced into the discharge channel.

(8) The cell analysis device according to (7), in which the separationcontrol part is a suction pressure application means provided in thedischarge channel.

(9) The cell analysis device according to (7), in which anultrafiltration membrane or a gel membrane for preventing the passage ofmolecules having the molecular size of the amplified product is disposedbetween the two-dimensional array chip and the three-dimensional porousbody.

(10) The cell analysis device according to (7), in which the separationcontrol part is a means for introducing a separation solvent or airbetween the nucleic acid capture part and the discharge channel.

(10-1) The cell analysis device according to (10), in which theseparation solvent is mineral oil.

(11) The cell analysis device according to (1) or (7), in which thenucleic acid capture part comprises a nucleic acid probe for capturing anucleic acid, and the nucleic acid probe comprises a nucleic acidcapture sequence which hybridizes with the nucleic acid extracted fromthe cell and a cell recognition sequence which is differentcorresponding to the respective cell capture part.

(12) A cell analysis method using the cell analysis device according to(1), including the steps of

filling the substrate with a solution containing cells; and

applying a negative pressure to the cell capture part, sucking thesolution containing the cells in direction to the substrate, andcapturing a single cell on the cell capture part.

(13) The cell analysis method according to (12), further including stepsof disrupting the single cell captured on the cell capture part in astate in which a negative pressure is applied to the cell capture part,and capturing the nucleic acid extracted from the cell by the nucleicacid capture part.

(14) The analysis method according to (13), further including a step ofsupplying to the nucleic acid capture part, a second nucleic acid probehaving a sequence which hybridizes with the nucleic acid captured by thenucleic acid capture part, and an enzyme and a substrate forcomplementary strand synthesis which uses the captured nucleic acid as atemplate to perform complementary strand synthesis.

(15) A cell analysis method using the cell analysis device according to(7), including the steps of

filling a solution containing cells on the two-dimensional array chip;and

applying a negative pressure to the cell capture part, sucking thesolution containing the cells from the discharge channel provided withthe three-dimensional porous body, and introducing a reagent for nucleicacid amplification to the nucleic acid capture part by suction in thesame manner as the solution containing the cells, and then, separatingthe nucleic acid capture part or the two-dimensional array chip from thethree-dimensional porous body until before the start of theamplification reaction or after the start and until before the end ofthe amplification reaction.

(15-1) The cell analysis method according to (15), further including astep for disrupting the single cell captured on the cell capture part ina state in which a negative pressure is applied to the cell capturepart, and capturing the nucleic acid extracted from the cell by thenucleic acid capture part.

(15-2) The analysis method according to (15-1), further including a stepfor supplying to the nucleic acid capture part, the second nucleic acidprobe having a sequence which hybridizes with the nucleic acid capturedby the nucleic acid capture part, and an enzyme and a substrate forcomplementary strand synthesis which uses the captured nucleic acid as atemplate to perform complementary strand synthesis.

(16) A cell analyzer including the cell analysis device according to anyone of (1) to (11), and a cell observation means (for example, afluorescence microscope).

Effect of the Invention

The cell analysis device, apparatus and cell analysis method of thepresent invention has succeeded in improving the cell capturingefficiency in the cell capture part, compared with the conventionaltechniques, while maintaining the nucleic acid capturing efficiency inthe nucleic acid capture part when performing nucleic acid capture fromcells. As a result, it becomes possible to prepare a sample for singlecell analysis in which the degree of separating the respective cells isaccurately made higher than that by using the conventional devices andthe like. Further, according to the cell analysis device, the apparatusand the cell analysis method of the present invention, it becomespossible to collect the amplification product with high efficiency whenperforming the amplification reaction inside the device.

Therefore, according to the cell analysis device, the apparatus and thecell analysis method of the present invention, it is possible toquantitatively analyze, with high efficiency and high accuracy, not onlythe expression level of genes as the average of tissues but also thecontents of the respective cells constituting tissues. By performingsingle cell analysis which measures, at the single cell level, genes(for example, mRNA) which are active in living tissue, it is possible toknow various phenomena occurring in vivo in detail including theinteraction between cells. This is expected to have a large effect onthe life sciences field, specifically, medicine, drug discovery,diagnosis, basic research of vital phenomena, and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a figure showing a basic configuration of the device describedin Patent Document 1 which is technology related to the presentinvention.

FIG. 2 is a figure showing an embodiment having the configuration of acell analysis device according to an aspect of the present invention.

FIG. 3 is a figure showing an embodiment having the configuration of thecell analysis device according to another aspect of the presentinvention.

FIG. 4 is a flow diagram of a cell analysis method using the cellanalysis device according to the present invention and a figure showingthe separation timing.

FIG. 5 is a figure showing an outline of the configuration of the devicemanufactured in Example 1-1.

FIG. 6 is a figure showing a process of treating a nucleic acid in theanalysis method using the cell analysis device according to the presentinvention.

FIG. 7 is a figure showing an outline of the configuration of the devicemanufactured in Example 1-4.

FIG. 8-1 is a figure showing an outline of the configuration of thedevice according to Example 2.

FIG. 8-2 is the continuation of FIG. 8-1.

FIG. 9 is a figure showing an outline of the configuration of the devicemanufactured in Example 2-2.

FIG. 10 is a figure showing an outline of the configuration of theapparatus according to Example 3.

MODES FOR CARRYING OUT THE INVENTION

In one aspect, when sucking the cells in the cell capture part of thetwo-dimensional array chip, the cell analysis device of the presentinvention prevents the adsorption of the cells on the two-dimensionalarray chip by sucking the cells in competition with a repulsive force(for example, gravity) in the direction which separates the cells fromthe substrate of the two-dimensional array chip. Specifically, the cellanalysis device of the present invention includes: a solutionintroduction channel for introducing a solution containing cells; asubstrate having a plurality of sets of a cell capture part and anucleic acid capture part, the cell capture part being in contact withthe solution introduction channel and having a concavity capable ofcapturing a single cell, and the nucleic acid capture part beingprovided in a corresponding manner to the concavity of the cell capturepart and capturing a nucleic acid extracted from the cell having beencaptured by the cell capture part; a discharge channel that is providedadjacent to the nucleic acid capture part of the substrate, anddischarges a solution of the nucleic acid capture part; and a pressurecontrol means provided in the discharge channel. The substrate has arepulsive force in a direction which separates the cells from thesubstrate. The pressure control means controls such that, when the cellis captured by the cell capture part, a force in the direction from thecell capture part to the nucleic acid capture part serves as a firstpressure that is larger than the repulsive force.

FIG. 2 is a figure showing an embodiment of the configuration of thecell analysis device according to the above aspect of the presentinvention, (a) shows a bottom view (plan view seen from the bottom), (b)shows a cross-sectional view in the 2A-2A′ cross section, and (c) showsa cross-sectional view in the 2B-2B′ cross section. Further, (d) is anenlarged view of the region 2C in the cross-sectional view (c).

The cell analysis device according to the present invention includes asubstrate (hereinafter, referred to as the two-dimensional array chip(213)) having sets of followings: a plurality of cell capture parts 203each of which can capture a single cell; and nucleic acid capture parts210 disposed corresponding to the respective cell capture parts andcapturing nucleic acids extracted from the cells captured by the cellcapture parts. A lower region 214 which holds a cell solution introducedfrom a cell introduction port 211 prior to sucking is provided adjacentto the two-dimensional array chip. An upper region 215 of the oppositeside is connected to an upper outlet 207 on the upper side. A pump or asyringe is connected to the upper outlet 207. By applying a negativepressure to the upper outlet 207, a negative pressure can be applied tothe nucleic acid capture parts 210 and the cell capture parts 203 on thetwo-dimensional array chip, and the cell solution within the region 214is sucked into the cell capture parts 203. As shown in enlargedcross-sectional view of (d), a force acts on the cells in an upwarddirection (the direction of arrow 216) due to the viscosity of thesolution during the sucking of the cell solution, whereas, gravity is inthe 217 direction, which is a direction opposite to that of the suctiondirection. The sedimentation of the cells caused by gravity operates ina direction to separate the cells from the two-dimensional array chip.Thus, the suction force for the cells operates only at the cell captureparts 203, which prevents cell adsorption to regions other than the cellcapture part on the two-dimensional array chip. While this embodimentutilizes gravity as the repulsive force in the direction which separatesthe cells from the two-dimensional array chip, use of other repulsiveforces will be described later.

The nucleic acid capture part 210 may be composed of a porous materialor beads (preferably, magnetic beads) and the like, and a nucleic acidprobe for capturing the nucleic acids extracted from the cells may beimmobilized. The nucleic acid probe includes a nucleic acid capturesequence which hybridizes with the nucleic acids extracted from thecells and a cell recognition sequence which is different correspondingto the respective cell capture parts. The single cell analysis ispossible by using the product in which the nucleic acids captured bysuch nucleic acid probe are amplified. The nucleic acid probe can beappropriately designed by a person skilled in the art in accordance withthe purpose of the analysis, the target to be analyzed (mRNA, genomicDNA, ncRNA, and the like), the configuration of the device to be used,the type of the amplification reaction, and the like.

The cell solution is introduced from the cell introduction port 211. Inthis case, the region 214 is filled with the cell solution in avertically inverted form with regard to the conventional example shownin FIG. 1, and the cell solution is sucked from the sample collectionport 212. Subsequently, a single cell is adsorbed on the cell captureparts 203 by applying a negative pressure to suck the solution from theupper outlet 207, which feature is the same as the device of theconventional example. However, the device according to the presentinvention is different from the device of the conventional example inthat the device is controlled to have the gravity work in a suitabledirection.

The suction force that causes the cells to approach the two-dimensionalarray chip during cell suction needs to be stronger than gravity whichis applied in the opposite direction. This strong suction is effective,particularly when the negative pressure which is applied to the upperoutlet is close to 1 atm, and the pressure on the inside of the region215 is set lower than the saturated vapor pressure of the cell solution.A continuous suction may be necessary to make the internal pressurelower than the saturated vapor pressure, thus, a continuous suction pumpsuch as a diaphragm pump may be more effective than a pump using asyringe having a limited suction volume. Another method for realizing astrong suction force which overcomes gravity is a method which disposesa three-dimensional porous member. When disposed in the region 215, thethree-dimensional porous member has the properties that the aqueoussolution contacted is quickly absorbed due to the capillary phenomenon,and, the absorbed aqueous solution can be discharged to the outside, inparticular, in a direction toward not being in contact with the nucleicacid capture part 210. By discharging the absorbed aqueous solution, thesolution absorption performance due to the capillary phenomenon of thethree-dimensional porous member can be maintained. By utilizing thecapillary phenomenon of the three-dimensional porous member, thesolution retained in the nucleic acid capture part 210 can be suckedmore rapidly, compared to the case when merely applying a negativepressure in the conventional example shown in FIG. 1, and further, theability that the cell capture parts 203 absorb cells can be improved.The embodiment using the three-dimensional porous member is explained inthe description of the configuration for efficiently collecting thenucleic acid amplification product when steps up to nucleic acidamplification are performed on the device.

The force applied to the cells in the direction which separates thecells from the two-dimensional array chip is not limited to gravity. Forexample, it is possible to apply a repulsive force to the cells in thevicinity of the two-dimensional array chip to prevent the adsorption ofcells on the two-dimensional array chip by subjecting thetwo-dimensional array chip (substrate) to a surface treatment having anegative charge relative to the two-dimensional array chip. Further,this can be realized even by coating the surface with a coating agentsuch as MPC polymer or polyethylene glycol.

Furthermore, it is also possible to apply a force in the direction whichseparates cells from the two-dimensional array chip bydielectrophoresis. This will be described in detail in Example 1-4.

Further, in another aspect, the three-dimensional porous member may beprovided adjacent to the two-dimensional array chip, by which the cellanalysis device of the present invention accelerates the suction ofcells due to the capillary phenomenon and reduces the influence ofsedimentation caused by gravity on the cells. The cell analysis deviceof the present invention, when provided with a three-dimensional porousmember, similarly, provides a means for separating the two-dimensionalarray chip and the three-dimensional porous member during theamplification reaction so that the amplification product amplified onthe two-dimensional array chip is not adsorbed on the three-dimensionalporous member. Specifically, the cell analysis device of the presentinvention includes a solution introduction channel for introducing asolution containing cells; a two-dimensional array chip having aplurality of cell capture parts and nucleic acid capture parts, the cellcapture parts being provided adjacent to the solution introductionchannel and each being capable of capturing a single cell, and each ofthe nucleic acid capture parts being provided corresponding to each cellcapture part of the plurality of cell capture parts and capturing anucleic acid extracted from the cell having been captured by the cellcapture part; a discharge channel having a three-dimensional porous bodywhich absorbs a solution retained in the nucleic acid capture part andfor discharging the solution; and a separation control part forcontrolling separation between the nucleic acid capture part and thedischarge channel. The separation control part performs separationcontrol such that, after the nucleic acid is captured by the nucleicacid capture part, a product amplified from the captured nucleic acid isnot introduced into the discharge channel.

FIG. 3 is a figure showing an embodiment of the configuration of thecell analysis device of the aforementioned aspect of the presentinvention, where (a) shows a bottom view (a plan view seen from thebottom), (b) shows a cross-sectional view in the 3A-3A′ cross section,and (c) and (d) show cross-sectional views in the 3B-3B′ cross section.

The cell analysis device according to another aspect of the presentinvention relates to the configuration characterized in providing ameans that can efficiently collect the amplification product from thenucleic acids extracted from cells to the outside. There is provided, inthe same manner as the above aspect, a two-dimensional array chip (313)having a plurality of cell capture parts 303 and nucleic acid captureparts 310, each of the cell capture parts being capable of capturing asingle cell, and each of the nucleic acid capture parts being providedcorresponding to the respective cell capture parts and being capable ofcapturing a nucleic acid extracted from the cells having been capturedon the cell capture parts. It is also the same as the above aspect thata lower region 314 which holds a cell solution introduced from a cellintroduction port 311 is disposed adjacent to the two-dimensional arraychip, and an upper region 315 of the opposite side is connected to anupper outlet 307. Furthermore, it is also the same that a pump or asyringe is connected to the upper outlet 307, a negative pressure can beapplied to the nucleic acid capture part 310 and the cell capture parts303 on the two-dimensional array chip by applying a negative pressure tothe upper outlet 307, and the cell solution within the region 314 issucked into the cell capture parts 303 are also similar. However, thepresent aspect is different in the point that a means for separating thethree-dimensional porous member from the two-dimensional array chip isprovided in a configuration for capturing cells by increasing thesuction force by disposing the three-dimensional porous member in theupper region. A “storage tank” (the space for introducing air or aseparation solvent such as a nonpolar solvent (for example, mineraloil)) 323 of FIG. 3, and a syringe 333 as a means for injecting air or aseparation solvent in the region between the three-dimensional porousmember and the two-dimensional array chip are provided as the separationmeans. In the present aspect, the two-dimensional array chip may beprepared with a resilient material (here, a PDMS resin), thus, byapplying a negative pressure for suction to the upper outlet 307, thetwo-dimensional array chip is bent by the negative pressure applicationto be in close contact with the three-dimensional porous member 319 asin the two-dimensional array chip shown by 313 in FIG. 3(C). Suction bythe capillary effect of the three-dimensional porous member becomespossible by such close contact. By stopping the application of negativepressure and injecting air or separation solvent between thethree-dimensional porous member 319 and the two-dimensional array chip313 from the “storage tank” 323 of the respective element, the migrationof a substance through the solution is blocked. The adsorption of DNA,which is the amplification product, on the inner wall of thethree-dimensional porous member can be prevented by this blocking.

Specifically, in order to maintain the state in which the numerous beadsconstituting the nucleic acid capture parts 310 in the two-dimensionalarray chip are packed, a membrane for preventing bead outflow(hydrophilic porous membrane) 318 is brought into close contact with theback surface of the two-dimensional array chip 313. The close contact ofthe three-dimensional porous member and the two-dimensional array chipmay be accurately performed by the close contact of a bead holdingmembrane 318 and the three-dimensional porous member. Here, the membranefor preventing bead outflow 318 functions integrally with thetwo-dimensional array chip 313, thus, the suction by the capillaryeffect is effective.

As the separation means, an actuator can be connected to thethree-dimensional porous member to change the distance between thethree-dimensional porous member and the two-dimensional array chip so asto separate the three-dimensional porous member and the two-dimensionalarray chip.

Furthermore, it is also possible to separate the three-dimensionalporous member and the two-dimensional array chip by providing anisolation membrane such that the amplification product does not reachthe surface of the three-dimensional porous member while keeping them inclose contact with each other. This step may be performed, for example,by disposing between the three-dimensional porous member and thetwo-dimensional array chip, an ultrafiltration membrane or a gelmembrane having a pore size which prevents the passing through ofmolecules (for example, DNA molecule) having the molecular size of theamplification product.

The three-dimensional porous member used in the present invention can bea material having pores ranging in size from several tens of nanometersto zero point several micrometers in a random manner, and having asurface hydrophilicity to the extent that an aqueous solution can beabsorbed by the capillary phenomenon produced by the presence of thepores. Specific examples may include a porous glass material, a glassfiber assembly (for example, a material in which glass fibers arearranged in the same direction and bundled), a glass bead assembly (theaverage particle diameter is preferably in the range of 0.1 to 30 μm)and the like. The three-dimensional porous member may preferably be noteasily deformed to lower the possibility that the absorbed solution mayunintentionally discharged and flows back to the nucleic acid capturepart 310 and the cell capture part 303. The hydrophilic surface of thethree-dimensional porous member can be characterized by either a watercontact angle measured using pure water being 90° or less, preferably80° or less, more preferably 50° or less, and most preferably 40° orless, or when dropping 1 μL of pure water on the three-dimensionalporous member having a volume sufficient to maintain 1 μL of liquid,absorbing the dropped droplets within 10 seconds, preferably within 5seconds, more preferably within 3 seconds, and most preferably within 1second. Note that, the expression that the droplets are absorbed by thethree-dimensional porous member means that the presence of the dropletscannot be confirmed visually when observed from a plane perpendicular tothe surface of the three-dimensional porous member.

Further, it may be preferable that the three-dimensional porous memberconstituting the solution holding part 319 is sufficiently larger inpore diameter than that of the pores belonging to the nucleic acidcapture part 310 which is composed of the porous membrane, beads, etc.For example, the average pore diameter may preferably be 0.2 μm or more,and especially 3 μm or more. If the pore diameter has such size, anexcessively large pressure loss does not occur when a gas such as airpasses through the solution holding part 319. Further, if the porediameter of the three-dimensional porous member is large as statedabove, a sufficient pressure difference can be produced between theupper and lower part of the nucleic acid capture part 310 when thenegative pressure is applied from the upper outlet 307.

Preferably, the flow channel including the solution holding part 319 andcommunicating with the upper outlet 307 also communicates with an intakeport 321 which opens to the outside of the apparatus through a pressureadjustment member 320. In the apparatus of FIG. 3, the intake port 321opens to the outside in the upper direction of the apparatus. In thecase where the intake port 321 is provided, when the negative pressureis applied from the upper outlet 307, the outside gas is taken in fromthe intake port 321 through the pressure adjustment member 320. Thisgenerates an airflow 322, which causes the solution absorbed in thesolution holding part 319 to be discharged. With the configurationincluding the intake port 321, the solution holding part 319 can absorbthe solution by the capillary phenomenon only when a negative pressureis applied from the upper outlet 307. That is, the solution holding part319 can absorb the solution again by the capillary phenomenon only forthe volume of the solution that has been discharged by the solutionholding part 319. Further, in the configuration without the intake port321, the solution may not reach the solution holding part 319 by onlyfilling the upper region 314 with the solution, so that absorption doesnot start; while on the other hand, if absorption starts, the absorptiondoes not stop until the solution holding part 319 is fully filled, whichmeans that the controllability is insufficient. In this case, thecontrollability can be compensated by providing the intake port 321. Itmay be preferable to further provide an air valve in the intake port321, as the controllability of the absorption of the solution is furtherimproved.

The pressure adjustment member 320, when a gas passes therethrough,preferably produces a pressure loss larger than the pressure lossproduced when a solution passes through the nucleic acid capture part310. By providing such pressure adjustment member 320, when a negativepressure is applied from the upper outlet 307, the negative pressure isapplied not only to the intake port 321, but also sufficiently to thesolution holding part 319, the nucleic acid capture part 310 and thecell capture part 302. As for the pressure adjustment member 320, thebalance may be good from the viewpoint of the viscosity differencebetween air and water if using a material having a pore smaller than thepore of the nucleic acid capture part 310, for example, a materialhaving an average pore diameter of about ⅕ to about 1/10, morepreferably about 1/7 to about 1/9, and especially, about ⅛ of the poreof the nucleic acid capture part 310, for example.

Note that, the present inventors already filed an international patentapplication for a cell analysis device provided with a three-dimensionalporous member (PCT/JP2015/077849 filed Sep. 30, 2015). The contentsdescribed in the specification, claims and drawings of the applicationare herein incorporated by reference as is.

FIG. 4 shows a flow diagram of the cell analysis method using the deviceaccording to the present invention. The timing at which thethree-dimensional porous member is separated from the two-dimensionalarray chip is explained with reference to the diagram.

After the cells are captured on the cell capture part 303 in the firststep as stated above, then in (Step 2), the cells are disrupted by alysis buffer and the like in a state in which a negative pressure isapplied from the upper outlet 307, and the nucleic acids extracted fromthe cells (for example, mRNA) are captured by the nucleic acid probeimmobilized on the nucleic acid capture part 310. In (Step 3), using thenucleic acids captured on the surface of the nucleic acid capture part310, a 1st strand of the complementary strand (for example, cDNA) of thecaptured nucleic acid (for example, mRNA) can be synthesized byintroducing a reagent containing an enzyme to the nucleic acid capturepart. In (Step 4), a 2nd strand can be synthesized by introducing asecond DNA probe corresponding to a gene to be measured and an enzymereagent to the nucleic acid capture part. Until this point, a reactionhas occurred at the surface of all of the nucleic acid capture parts,and necessary reaction products may be immobilized on the surface. Inthe subsequent amplification reaction (for example, PCR), theamplification product is released from the surface, and collected fromthe device as a final product. At this time, in order to prevent theamplification product from being adsorbed on the inner wall of thethree-dimensional porous member, the suction force is turned off afterthe reagent containing the enzyme and the substrate necessary for theamplification reaction are introduced to the nucleic acid capture partby suction, and air or a separation solvent for separation is injectedbetween the three-dimensional porous member and the two-dimensionalarray chip from a “storage tank” 323 by pressing the syringe 333.Therefore, the amplification product is blocked from migrating fromwithin the two-dimensional array chip to the three-dimensional porousmember, and the adsorption can be prevented. During amplification, theamplification product spreads out from within the two-dimensional arraychip by diffusing into the region 314.

The timing of the separation by the injection of air or the separationsolvent may also be the timing before the amplification reaction (forexample, PCR cycle reaction) after reagent introduction. However, it maybe better to separate the three-dimensional porous member and thetwo-dimensional array chip after completing several cycles of theamplification reaction prior to the migration from the nucleic acidcapture part to the three-dimensional porous member by diffusion. Theinfluence of the migrating of the solution due to temperature variationcan be suppressed to a minimum by performing the separation at suchtiming.

After the completion of the amplification step, the amplificationproduct is collected from the sample collection port 312 by suction. Atthis time, the cell introduction port 311 is opened, and the solutionmigrates from 311 toward 312. DNA strands (especially, short DNAstrands) of lengths which are not necessary for analysis are containedin the obtained amplification product, thus, in (Step 7), purificationis conducted to remove these DNA strands, and the sequence analysis isperformed by a next-generation sequencer in (Step 8). Finally, obtainingthe results of the single cell analysis by rearranging the sequencesobtained in the tag sequences corresponding to the cell capturepositions (Step 9) may be similar to the conventional example. Notethat, the amplification of the nucleic acid may preferably be PCRamplification, but not limited thereto, and it is possible to use otheramplification methods such as rolling circle amplification (RCA)reaction, NASBA method, and the LAMP method. These other amplificationmethods are well-known in the technical field, thus, a person skilled inthe art could appropriately select a nucleic acid probe and a reagent(s)to be used.

Further, when separating the three-dimensional porous member from thetwo-dimensional array chip with an actuator, the separation may also beperformed at the same timing as stated above.

Furthermore, in the separation method using the ultrafiltration membraneor the gel membrane, the migration of the amplification product to theporous member may be blocked at all times, thus, it is not necessary toset the timing of the separation. Herein, the amount of migration of thenucleic acid of the amplification product can be reduced to 1/10 or lessin a state in which there is no negative pressure application by settingthe pore size of the ultrafiltration membrane or the gel membrane to 1to 10 nm, for example, 5 nm (30 kDa).

EXAMPLES

The present invention will be explained in greater detail below usingexamples; however, the present invention is not limited to theseexamples.

(Example 1-1) Manufacture of the Device

FIG. 5 is a figure showing an outline of the configuration of the devicemanufactured in the present example, where (a) shows a bottom view (planview seen from below), and (b) shows a cross-sectional view in the5A-5A′ cross section. The device 500 includes a plurality of reactionchambers 524 each of which includes a two-dimensional array chip 513arranged thereon, the two-dimensional array chip 513 includes sets ofthe plurality of cell capture parts 503 and nucleic acid capture parts510 corresponding to the respective cell capture parts 503. A cellsolution is caused to flow through a common flow channel 525 from a cellintroduction port 511 toward a sample collection port 512, and thensupplied on the two-dimensional array chip 513. The chip is thus filledwith the cell solution. Separate inlets 527 may be used when separatelyintroducing a reagent to the chips, specifically, for example, whenintroducing a reagent containing a primer having a tag sequence inpurpose of identifying the chip.

A resin chip (square shape in which one side is 1.125 mm) made ofdimethylpolysiloxane (PDMS) obtained by injection molding was used inthe manufacture of the two-dimensional array chip 513, and athrough-hole having a diameter in the range of approximately 3 to 10 μmwhich is smaller than the cells was formed to make the cell capture part503. Note that, the manufacture of the two-dimensional array chip may beperformed using a resin chip obtained by the injection molding usinganother resin (polycarbonate, cyclic polyolefin, polypropylene, and thelike), or may be performed using a nanoimprint technology or asemiconductor process. The material used in the manufacturing of thetwo-dimensional array chip may preferably be a hydrophobic material, bywhich the adsorption of the cells, the reagent, and the like on thearray chip can be reduced. By subjecting the surface of the substrate ofsuch two-dimensional array chip to a treatment to prevent the adsorptionof cells, it is also possible to apply a repulsive force to the cells(refer to Example 1-3).

A cylindrical space having a diameter of several tens of μm (forexample, 10 to 50 μm) is provided directly below the cell capture part503, and then the cylindrical space is filled with magnetic beads onwhich a probe for a nucleic acid capture is immobilized to thusconstitute the nucleic acid capture part 510. The filling with themagnetic beads may be performed separately using an inkjet printer head.While the chip is vertically inverted, the nucleic acid capture part 510is separately filled with 2 nL of a solution of the beads on whichdifferent sequences are immobilized according to each regions. Themagnetic beads having a diameter of 1 μm may be suspended at a numberdensity of 5×10⁹/mL in the bead solution used for filling. Streptavidinis immobilized on the magnetic beads, and a DNA probe modified with 5′biotin is immobilized through streptavidin. The beads are made to have adiameter of several μm or less (for example, 1 μm or less) to increasethe number of magnetic beads to be filled, and thereby improving thenucleic acid capturing efficiency. A membrane for preventing beadoutflow 518 (porous membrane made of resin having a pore size of 0.8 μm:Isopore membrane, manufactured by Millipore Corporation) having asmaller pore diameter than the bead diameter is brought into closecontact with the two-dimensional array chip 513 such that the filledmagnetic beads do not flow out.

In the present example, gravity is used as the repulsive force, and thecells are prevented from contacting with and adsorbing on the regionsother than the cell capture part on the two-dimensional array chip dueto the sedimentation of cells with gravity. In the present example,gravity operates in the direction which separates the cells from thetwo-dimensional array chip, thus, it is necessary to suck the cells inorder to overcome gravity. However, the nucleic acid capture part 510 ofthe two-dimensional array chip 513 is filled with magnetic microbeads,thus, by only applying a pressure difference to the upper and lowerparts of the two-dimensional array chip 513 using the syringe, the cellsolution does not flow at a sufficient flow rate and a sufficientsuction force cannot be obtained. Therefore, in the present example, asa more preferred embodiment, the suction method is performed not byusing the syringe, but by connecting the diaphragm pump to the upperoutlet, which keeps the pressure in the region of the upper flow channel526 at or below the saturated vapor pressure of an aqueous solution soas to evaporate and rapidly discharge the solution which reached theback surface of the two-dimensional array chip. Evaporating the solutionin this way promotes the capillary effect of the nucleic acid capturepart to thereby realize high-speed suction can be performed and tocapture the cells against gravity.

(Example 1-2) Single Cell Analysis Method Using the Device

FIG. 4 is a flow diagram showing an outline of the single cell analysismethod using the device of the present invention manufactured in Example1-1 shown in FIG. 5. In the present example, (Step 1) cell capturing,(Step 2) cell lysis (disruption), (Step 3) complementary strand (1ststrand) synthesis (for example, cDNA synthesis), (Step 4) capturednucleic acid degradation (for example, mRNA degradation), (Step 5) 2ndstrand synthesis, and (Step 6) the amplification reaction are performedin the device. In the reaction until (Step 5), the reaction product maybe immobilized on the nucleic acid capture part 510, and the reaction isa solid phase reaction. In (Step 6), the amplification product releasedfrom the two-dimensional array chip diffuses into a reaction reagent inthe reaction chamber 524. After completion of the amplificationreaction, the reagent containing the amplification product may becollected from the sample collection port 512 by suction.

Further, after completion of the complementary strand (1st strand)synthesis (for example, cDNA synthesis) of (Step 4), the two-dimensionalarray chip may be removed from the device and the chip may be submergedin the tube containing the reagent so as to collect the beads in thesolution (Step 5). After that, the subsequent reactions may be performedin the tube (outside of the device).

(Step 7) is a purification step which removes by-products duringamplification, which are unnecessary for nucleic acid sequence analysis.Furthermore, (Step 8) is a sequence analysis step using anext-generation sequencer, and any sequence analysis platform may beused as long as it is a sequencer with a high parallelism. The finalstep (Step 9) is data analysis step, which is a step for summarizing thesequence analysis results for the cell identification tags and chiptags, and for constructing the genetic analysis data of each singlecell.

First, the step for capturing the cells in the cell solution by usingthe cell capture parts 503 on the two-dimensional array chip 513 isperformed. To perform the step, the two-dimensional array chip 513 isfilled with the cell solution by introducing the cell solution from thecell introduction port 511 and sucking the cell solution from the samplecollection port 512. Next, by applying a negative pressure to the loweroutlet 507, the cell solution passes through the cell capture parts 503and the nucleic acid capture part 510 on the two-dimensional array chip513, and the cells are captured on the cell capture parts 503. 501 ofFIG. 5(a) shows the cells which are captured on the cell capture part.At this time, high speed suction can be performed by continuousexhaustion with the diaphragm pump and the capillary effect of thenucleic acid capture part to thereby overcome the sedimentation withgravity. The suction time may be within 1 minute per 1 μL. At this time,the adsorption of cells on the two-dimensional array chip due to thesedimentation is 10% or less.

To disrupt the cells, the two-dimensional array chip 513 is filled witha lysis buffer by introducing the lysis buffer from the cellintroduction port 511 and sucking the lysis buffer from the samplecollection port 512. Then, a negative pressure is applied to the loweroutlet 507 so as to obtain the necessary suction rate. The appliednegative pressure is made to the same as the pressure during the cellsuction. Note that, to complete the cell suction, it may be effective tointroduce a PBS buffer of approximately 1 μL before introducing thelysis buffer.

Next, the process proceeded to the step of complementary strand (1ststrand) synthesis (for example, reverse transcription, cDNA synthesis).To achieve a necessary introduction rate with a reagent mix for thecomplementary strand synthesis (reverse transcription), the strength ofthe negative pressure is reset. After setting, the reagent is introducedfrom the cell introduction port 511 to fill the two-dimensional arraychip 513 with the reagent. Then, to proceed with the complementarystrand synthesis reaction (reverse transcription reaction), thetemperature of the device is increased and maintained only for the timenecessary for the reaction. Furthermore, the temperature is increased to85° C. in order to inactivate the reagent (for example, reversetranscriptase). A washing buffer is introduced from the cellintroduction port 511 to wash out and discharge unnecessary washingsolution, and the negative pressure is reset to introduce and dischargethe washing buffer in the nucleic acid capture part. The degradation ofthe captured nucleic acids (mRNA degradation) and the 2nd strandsynthesis being the next steps are almost the same as the step of thecomplementary strand (1st strand) synthesis (reverse transcription).

The final step is the amplification reaction. The amplification reactionreagent mix is introduced to the cell introduction port 511, and anegative pressure is set so as to introduce the reagent mix to thenucleic acid capture part 510 at the necessary solution rate. Thetemperature cycle is started to perform the amplification reaction(especially, PCR), and the pressure setting is changed such that thesyringe is simultaneously operated when the application of the negativepressure is stopped in the first to several cycles. After that, thetemperature cycle is repeated until the necessary concentration ofamplification product is achieved. Then, a negative pressure is appliedto the lower outlet 507 to suck and collect the amplification product,and the collected amplification product solution is discharged in thetube.

Next, the preparation procedure of a sample for sequencing using thetwo-dimensional array chip 513 will be described in detail. FIG. 6 showshow the sample is prepared by treating the nucleic acid (for example,mRNA) captured by the nucleic acid capture part 510 of the device of thepresent example. This step can be divided into the disruption of thecells, after cell capturing (Step 1), the capturing of nucleic acid(mRNA) (Step 2), the synthesis of a complementary strand (1st strand)(for example, cDNA) (Step 3), the degradation of the captured nucleicacid (for example, mRNA) (Step 4), and nucleic acid amplification (PCR)and synthesis of 2nd strands into which known terminal sequencesnecessary for sequencing have been introduced (Step 5), and further, thesteps of nucleic acid amplification (Step 6-1) and (Step 6-2).

After washing the cells with 500 μL of 1×PBS buffer without damaging thecells, 1000 cells suspended in 10 μL of 1×PBS buffer cooled to 4° C. areintroduced from the cell introduction port 511, and sucked from theupper outlet 512 so that the reaction chamber 524 is filled with thesolution. Therefore, the two-dimensional array chip 513 is filled withPBS buffer containing cells. Next, a negative pressure of 0.95 atm isapplied to the lower outlet 507 by, for example, the diaphragm pump tosuck the solution such that the solution flows through the cell capturepart 503 toward the upper flow channel 526. The cells migrate along withthe flow of the solution to reach the cell capture part 503. Since thediameter of the opening of the cell capture part 503 is smaller than thediameter of the cells, the cells are captured thereon. The capturedcells serve as stoppers for the solution flow, thus, the solution flowmoves to a cell capture part 503 that has yet to capture any cells.Therefore, the remaining cells migrate to capture parts that have yet tocapture any cells and are captured.

When a desired number of cells have been captured, excess cells whichhave not been captured and the PBS buffer are discharged from the samplecollection port 512. Next, after the lysis buffer (for example, asurfactant such as Tween 20) flowed from the cell introduction port 511toward the sample collection port 512, and the two-dimensional arraychip 513 is filled with the buffer, the solution is immediately suckedby applying a negative pressure to the lower outlet 507. All of thesolutions hereinafter described pass through the nucleic acid capturepart 510 of the two-dimensional array chip 513 by the same manner. Atthis time, as the membrane for preventing bead outflow 518 is a flowchannel constituted from the porous material having a diameter of 0.8μm, and has a large pressure loss. Thus, it may be easy for the lysisbuffer to continuously and slowly flow for about 5 minutes through thecell capture part 503 toward the upper flow channel 526 by using suchmembrane for preventing bead outflow 518.

The captured cells 501 are disrupted by the lysis buffer, and thenucleic acid 606 (for example, mRNA) exits to the outside of the cell.However, the cell solution flowing in the vicinity of the cell capturepart 503 continues to flow so as to be sucked into the pore constitutingthe cell capture part 503, thus, the nucleic acid 606 (for example,mRNA) reaches the nucleic acid capture part 510 through the cell capturepart 503 without diffusing to the periphery. Therefore, the disruptionof the cells and the capturing of the nucleic acid 606 (for example,mRNA) by a first DNA probe 601 immobilized on the bead of the nucleicacid capture part 510 are performed simultaneously by the introductionof the lysis buffer. The state is shown in Step 2 of FIG. 6.

Herein, in order to incorporate into the genetic analysis data theposition information on the two-dimensional array chip 513 in which thecells are captured, i.e., the position coordinates of the cell capturepart 503 arranged in a lattice-shape as shown in FIG. 5(a), a first DNAprobe 601 in which a cell recognition sequence 602 having a sequencewhich is different in a corresponding manner with the respective cellcapture parts 503 is immobilized on the bead surface of the nucleic acidcapture part 510. In FIG. 6, the shaded parts at the left end of thediagram of each step show a wall surface to be immobilized, herein, thebead surface. The first DNA probe 601 has a sequence (for example, whencapturing mRNA, a poly (T) sequence) complementary to the nucleic acidto be captured in the 3′ terminal region, thus, the nucleic acid 606(for example, mRNA) may be captured by hybridizing the probe with thesequence of the nucleic acid to be captured (for example, by hybridizingwith the poly (A) sequence at the 3′ terminal of mRNA). Further, auniversal primer (604, Reverse) for the amplification reaction may beprovided on the 5′ terminal side.

In the present example, the first DNA probe 601 contains, from the 5′terminal, in the order of a universal primer (604, Reverse) foramplification having approximately 30 bases, a cell recognition sequence(602) having approximately 7 bases, and an oligo (dT) sequence havingapproximately 18 bases+a VN sequence having 2 bases. In the presentexample, a poly (T) sequence is used in a part of the DNA probe 601 forcapturing in order to analyze the mRNA; however, a part of the sequencecomplementary to the sequence of the nucleic acid to be analyzed, or arandom sequence can be used in place of the poly (T) sequence in orderto perform microRNA and genome analysis. The sequence of such probe forcapturing can be appropriately designed by a person skilled in the artbased on conventional techniques.

Next, as shown in Step 3 of FIG. 6, the nucleic acid (mRNA) 606 capturedby the first DNA probe 601 is used as a template to synthesize the 1ststrand 607. In the present example, to synthesize the 1st cDNA from themRNA, as a reagent for synthesizing the 1st strand, 58.5 μL of 10 mMTris Buffer (pH=8.0) containing 0.1% Tween 20, 4 μL of 10 mM dNTP, 225μL of 5×RT Buffer (SuperScript III, Invitrogen Corporation), 4 μL of0.1M DTT, 4 μL of RNaseOUT (Invitrogen Corporation), and 4 μL ofSuperscript III (reverse transcriptase, Invitrogen Corporation) aremixed, and introduced from the cell introduction port 511 in the samemanner as the previous step. While the aforementioned solution flowingvery slowly from the reaction chamber 524 to the upper flow channel 526in a state in which the gaps between the packed beads are filled with asolution containing a synthesis reagent (for example, reversetranscriptase) and a synthesis substrate, the temperature of thesolution was slowly raised to 50° C. and the complementary strandsynthesis reaction (1st strand synthesis reaction) was performed forabout 50 minutes. As a result, the nucleic acid (for example, cDNA)immobilized on the surface of the plurality of beads for each cell isobtained as a library. Such libraries should be referred to as singlecell nucleic acid (cDNA) library arrays, which are fundamentallydifferent than the conventional normalized nucleic acid (cDNA) librariesobtained from many cells.

After the 1st strand 607 was synthesized, the entire device wasmaintained at 85° C. for 1.5 minutes to inactivate the synthesis reagent(for example, reverse transcriptase). After cooling to 4° C., 0.2 mL of10 mM Tris Buffer (pH=8.0) containing RNase and 0.1% Tween 20, forexample, was injected from the upper inlet 511 and sucked from the upperoutlet 512 to fill the reaction chamber 524 with the solution. Afterthat, the solution was discharged from the lower outlet 507 and thebuffer in the reaction chamber 524 was removed from the upper outlet512. By repeating this step for five times, the captured nucleic acid(mRNA) was degraded and the substances and the degraded substancesremaining in the nucleic acid capture part were removed and washed out.Furthermore, washing was performed for five times in a similar mannerwith a liquid containing an alkaline denaturing agent and a washingliquid. A nucleic acid library array corresponding to all of the cells,for example, a cDNA library array can be constructed by the process upto this point for each of the captured cells as shown in Step 4 of FIG.6.

Next, 69 μL of sterile water, 10 μL of 10× Ex Taq Buffer (TaKaRa BioCorporation), 100 μL of 2.5 mM dNTP Mix, and 1 μL of ExTaq Hot startversion (TaKaRa Bio Corporation) to which 10 μm of each of the universalsequences for amplification (Reverse) 609 is added were mixed, and thismixed reagent was introduced from the upper inlet 511 to the nucleicacid capture part 510 in the same manner as in the previous step. Themethod for separately introducing a second DNA probe 608 with a chipidentification tag 610 will be described later. Then, after dissolvingthe secondary structure of the nucleic acid at 95° C. for 3 minutes, thegene-specific sequence 611 of the primer was annealed with the 1ststrand as a template at 44° C. for 2 minutes. Step 5 of FIG. 6 shows thestate in which the second DNA probe 608 is hybridized to the 1st strand,and the 2nd strand 612 is synthesized. The complementary strandelongation reaction is completed by increasing the temperature to 72° C.for 6 minutes.

The introduction of the second DNA probe 608 to the reaction chamber 524is performed as follows. First, mineral oil is introduced from the cellintroduction port 511, and discharged from the upper outlet 512. Next,the buffer solution containing the reagent flows through the upper flowchannel 526 toward the separate inlets 527, and excess mineral oilwithin the reaction chamber is discharged from the separate inlets 527.In this manner, the regions filled with the buffer solution in thereaction chamber 524 at the lower portion of the two-dimensional arraychip 513 are separated by the mineral oil. Such separation occursbecause the inner wall of the reaction chamber 524 is hydrophilic, whileon the other hand, a surface treatment is performed such that the region528 between the reaction chambers is hydrophobic. Further, it ispreferable that the 1st strand is firmly immobilized to the beads (forexample, by biotin-avidin bonding) in order to maintain high reactionefficiency. After completion of the separation of the reaction chamber524, a buffer solution containing the second DNA probe having therespective different chip identification sequences is introduced fromthe separate inlet 527, and the buffer solution is discharged from theupper outlet 512 through the upper flow channel 526. The vicinity of thenucleic acid capture part 510 is thereby filled with a different secondDNA probe in each reaction chamber 524, and this probe hybridizes to the1st strand. After completion of the synthesis of the 2nd strand, a largeamount of the buffer solution flows from the cell introduction port 511toward the sample collection port 512, and the mineral oil in the region528 is washed away. As the flow channels only need to be connected, somemineral oil may remain in the region 528.

The final step is the amplification reaction by the universal primer asshown in Step 6-1 and Step 6-2 of FIG. 6. In the present example, a PCRreaction was performed, and 49 μL of sterile water, 10 μL of 10× HighFidelity PCR Buffer (Invitrogen Corporation), 10 μL of 2.5 mM dNTP mix,4 μL of 50 mM MgSO₄, 10 μL of 10 μm universal primer for PCRamplification (Forward), 10 μL of 10 μm PCR the universal primersequence for amplification (Reverse), and 1.5 μL of Platinum TaqPolymerase High Fidelity (Invitrogen Corporation) was mixed to preparethe reagent. Then, the reagent was introduced from the cell introductionport 511 in the same manner as the previous step. Subsequently, theentire device was maintained at 94° C. for 30 seconds, and a 3-stepprocess of 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for30 seconds was repeated 40 times. Then, at the end, the entire devicewas maintained at 68° C. for 3 minutes. After that, the entire devicewas cooled to 4° C. to perform the amplification step. This reaction isa universal reaction, and makes it possible to ensure uniformamplification efficiency among the chips by performing the amplificationreaction under the universal conditions for all of the chips. Forexample, a PCR Purification Kit (QIAGEN Inc.) is used for purificationin order to collect the amplification product solution accumulated inthe solution and to remove the remaining reagents contained in thesolution, such as free universal primer sequences for amplification(forward/reverse) and enzymes.

The obtained amplification product 615 is a sequence for which sequenceanalysis is possible, and is called a sequencing library. Even if anamplification bias occurs between the genes or molecules in this step,the amplification bias can be corrected with the use of the cellrecognition tag after acquisition of the sequencing data, thus, highlyaccurate quantification data can be obtained. The gene expression levelsper the cell identification sequences and the chip identificationsequences can be obtained by performing sequence analysis of thissequencing library. Namely, the simultaneous analysis regarding a numberof cells less than or equal to the number of the types of cellidentification sequences simultaneously introduced into the devicemultiplied by the type of chip identification sequences is possible. Theanalysis of the number of cells which is significantly larger than thenumber of cell identification sequences introduced in advance in thetwo-dimensional array chip is thereby possible.

Note that, the present inventors already filed an international patentapplication for a cell analysis method enabling simultaneous performanceof the genetic analysis of a single cell and the comprehensive geneticanalysis of a cell population containing the cell (PCT/JP2014/073753filed Sep. 9, 2014). The contents described in the specification, claimsand drawings of the application are herein incorporated by reference asis.

(Example 1-3) Application Method of Repulsive Force Other than Gravity(Surface Treatment)

The two-dimensional array chip can be prepared using PDMS resin. Thesurface of the two-dimensional array chip (substrate) made of a PDMSresin is coated before injecting the beads constituting the nucleic acidcapture part. Thus, a repulsive force acts on the cells in a directionwhich separates the cells from the two-dimensional array chip(substrate) in the vicinity of the two-dimensional array chip surface,and it is possible to prevent the adsorption of the cells on the chipsurface. In the present example, by coating an MPC(2-methacryloyloxyethyl phosphorylcholine) polymer (Lipidure (RegisteredTrademark)-CM5206 manufactured by NOF Corporation), the adsorption rateof the cells can be reduced to 1/10 or less. As the coating method, a0.5 w % ethanol solution was prepared, 2 μL per one chip was dropped ona PDMS resin membrane, the chip was covered with the solution, leftstanding for 5 minutes, and the chip was collected. The structure of thedevice is the same as FIG. 5, and the cells were hardly adsorbed and 95%or more of the cells were collected in all of the chips. Other than MPCpolymer, a PEG (polyethylene glycol)-based coating agent (Blockmastermanufactured by JSR Life Sciences Corporation) and the like may be usedas the coating agent, and any coating agent may be used.

(Example 1-4) Application Method of Repulsive Forces Other than Gravity(Configuration Using Dielectrophoresis)

FIG. 7 shows the configuration using dielectrophoresis to apply arepulsive force to the cells so as to reduce the adsorption of cells onthe two-dimensional array chip. The differences from the basicconfiguration shown in FIG. 5 are that an electrode fordielectrophoresis is vertically disposed, that the beads of the nucleicacid capture part are configured by metallic gold microparticles insteadof magnetic beads, and that a high frequency power supply is disposed.

The electrode configuration for preventing the adsorption of cells is asfollows. An upper electrode 729 is disposed in an upper flow channel726, and is a flat substrate made of platinum. A 1.1 mm square electrodedisposed directly above the two-dimensional array chip and havingapproximately the same size as the two-dimensional array chip isconnected in series in the same number as the number of chips. Further,the thickness of the electrode is set to 0.1 mm Because the appliedvoltage is low in the usage method of the present example, thepossibility of corrosion is low, and the material may be a metal otherthan platinum. Further, in a lower electrode 730, a wire having adiameter of 0.1 mm is disposed in a lattice-shape and an electrodestructure is molded by ultrasonic welding. Furthermore, as shown in FIG.7 (a), the wire is disposed so as to be positioned between the cellcapture parts. Further, the nucleic acid capture part 731 is packed withgold microparticles having a diameter of 400 nm, and uses a thiol groupon the surface to immobilize the DNA probe. By making the nucleic acidcapture part from gold microparticles (gold colloid), a suction forceacts toward the cell capture part by dielectric coupling. Note that, theAC voltage applied to both electrodes from a high frequency power supply732 is a sine wave of 10 Vpp at several kHz and has a frequency lowerthan the frequency which reverses from a dielectric attractive force toa dielectric repulsive force. As a matter of course, the frequency maybe higher than several MHz (the frequency is different depending on sizeof the cells and the dielectric constant) to make the dielectric forceas a repulsive force and use antagonistically with the suction force.

Example 2

FIG. 8 is a figure showing an outline of the configuration of the deviceaccording to another aspect of the present invention; (a) shows a topview, and (b) shows the configuration at the time of cell solutionsuction in a cross-sectional view at the 8A-8A′ cross section. Further,FIG. 8(c) shows a cross-sectional view when the three-dimensional porousmember and the two-dimensional array chip are separated during theamplification reaction. The device of the present example is aconfiguration which does not arrange gravity and the suction force inthe opposite direction as in Example 1 (may be placed in the oppositedirection), increases the suction force by using the three-dimensionalporous member, and reduces the influence of sedimentation with gravityto a minimum. At this time, the device structure and the operationmethod provided with a means for separating the three-dimensional porousmember from the two-dimensional array chip in order to prevent theadsorption of the amplification product to the inside of thethree-dimensional porous member disposed in a solution holding part 819which is the problem to be solved will be described focusing on thepoints of difference from Example 1.

FIG. 8 (b) is a cross-sectional view of the device while performing cellcapturing. The two-dimensional array chip 813 is made of a PDMS resinhaving elasticity, and uses a porous membrane made of resin having apore size of 0.8 μm (Isopore membrane, manufactured by MilliporeCorporation) as a membrane for preventing bead outflow 818 forpreventing the outflow of the beads which form a nucleic acid capturepart 810.

The device 800 includes a plurality of reaction chambers 824, each ofwhich includes a two-dimensional array chip 813 provided thereon, thetwo-dimensional array chip 813 having a plurality of cell capture parts803 and nucleic acid capture parts 810 corresponding to the respectivecell capture parts 803. A cell solution is caused to flow through acommon flow channel 825 from a cell introduction port 811 toward asample collection port 812, and then supplied on the two-dimensionalarray chip 813. The chip is thus filled with the cell solution. Separateinlets 827 are used when separate reagents are to be introduced into thechips, specifically, for example, when introducing a reagent containinga primer having a tag sequence for identifying a chip and the like.

A porous shirasu sintered member (Shirasu porous glass: SPG membrane,manufactured by SPG Technology Co., Ltd) which is a three-dimensionalporous member is disposed in a lower flow channel 837 of the nucleicacid capture part 810, and is made as the solution holding part 819. Byusing this three-dimensional porous member, sucking at a high speed ispossible, and the influence of sedimentation with gravity can beeliminated. The lower flow channel 837 is in communication with a loweroutlet 838 and an intake port 835 which takes in the air, and isprovided with a pressure adjusting filter 836 (in the present example,the porous membrane made of resin (Isopore membrane, manufactured byMillipore Corporation) having a pore diameter of 0.1 μm is used inconsideration of the difference in viscosity between water and air) andan air valve 834 in the vicinity of the intake port 835. When capturingthe cell in the cell capture part 803, the two-dimensional array chip813 is filled with the cell solution, and then a negative pressure isapplied by the syringe pump connected to the lower outlet 838 with theair valve 834 being open. Another pump such as a diaphragm pump can beused in place of the syringe pump.

The two-dimensional array chip is bent by applying a negative pressureto the lower outlet 838, and the membrane for preventing bead outflow818 and the three-dimensional porous member (819) are in close contactwith each other. Thus, the capillary effect of the three-dimensionalporous member can be used to suck the cell solution at a high speed.

A high speed suction method using the capillary effect will be describedbelow. In the device shown in FIG. 8, if the pressure relationships inposition A (the upper part of the two-dimensional array chip 813),position B (the lower flow channel 837 between the solution holding part819 and the pressure adjusting filter 836), position C (between thetwo-dimensional array chip 813 and the solution holding part 819), andposition D (the lower flow channel 837 closer to the lower outlet 838than the solution holding part 819) shown in the figure are set so as tosatisfy the magnitude relationship of A>>B>C>D, the nucleic acid capturepart 810 can be filled with the solution, and furthermore, the solutioncan come into contact with the solution holding part 819, and thecapillary phenomenon is produced in the three-dimensional porous memberconstituting the solution holding part 819 to generate a large suctionforce. In the case where the pore size of the three-dimensional porousmember is 10 μm, for example, it can be calculated that the suctionforce due to this capillary phenomenon produces a suction force of about100 kPa in the cell capture part 803 (assumed to be a circular shapehaving a diameter of 5 μm) in the configuration of the present example.Thus, the cells can be sucked with substantially no influence on thesedimentation with gravity.

The air taken in from the intake port 835 passes through the solutionholding part 819 due to the pressure difference between position B,position C and position D, and an aqueous solution absorbed by thesolution holding part 819 is thereby discharged toward position D. Thesuction force due to the capillary phenomenon of the solution holdingpart 819 is recovered by this discharge, thus, as long as the suctionfrom the lower outlet 838 continues, the suction force of the solutionholding part 819 is maintained. On the one hand, if the suction from thelower outlet 838 is stopped, the inner wall of the pore of thethree-dimensional porous member constituting the solution holding part819 is rapidly filled with the aqueous solution, and the suction forcemay be lost.

Next, the separation method of the three-dimensional porous member fromthe two-dimensional array chip will be described. After filling thereaction chamber 824 with the reagent for the amplification reaction(PCR reaction) in the same manner as Example 1, the amplificationreagent (PCR reagent) is introduced to the nucleic acid capture part byapplying a negative pressure to the lower outlet 838. Then, the syringe833 is pressed, and the mineral oil accumulated in the “storage tank”823 is slowly injected between the membrane for preventing bead outflow818 and the three-dimensional porous member over approximately 10seconds. At this time, as the three-dimensional porous member is highlyhydrophilic, most of the mineral oil which is a nonpolar solvent doesnot penetrate into the porous member. In this way, as shown in FIG.8(c), the amplification reagent (PCR reagent) solution which remained inthe three-dimensional porous member can be isolated from theamplification reagent (PCR reagent) in the nucleic acid capture part inthe two-dimensional array chip. When air is used in place of the mineraloil, the air enters into the three-dimensional porous member, and theamplification reagent (PCR reagent) solution can be isolated from thetwo-dimensional array chip in the same manner.

(Example 2-2) Separation Due to the Moving Mechanism of theThree-Dimensional Porous Member

In Example 2-1, a nonpolar solvent or oil for separation is injected;however, in the present example, it is also possible to separate thesolution by increasing the interval between the three-dimensional porousmember and the two-dimensional array chip at an appropriate timing.Specifically, a moving mechanism may be inserted directly below thethree-dimensional porous member. The present example shows an exampleusing an air pressure; however, a moving mechanism such as a servomechanism may also be used.

The configuration for separating the two-dimensional array chip from thethree-dimensional porous member by the air pressure is shown in FIG. 9.939 is a polyethylene resin air bag. The air bag has a size ofapproximately 1×1 mm similar to the two-dimensional array chip and isdesigned such that the thickness of the air bag changes to about 0.1 to0.3 mm by injecting air into the air bag with using a pump such as asyringe 940. When the syringe 940 is pressed to inject air, thetwo-dimensional array chip and the three-dimensional porous member arein close contact with each other and the suction of the solution due tothe capillary effect becomes possible. The three-dimensional porousmember is separated from the two-dimensional array chip by utilizing thefact that the internal pressure of air bag 939 decreases and thethickness thereof becomes thinner by drawing the syringe 940.

It may be possible to prevent the adsorption of the amplificationproduct to the inner wall of the three-dimensional porous member byincreasing the internal pressure of air bag before the amplificationreagent (PCR reagent) introduction and by decreasing the internalpressure of the air bag after the start of the amplification reaction(for example, a PCR cycle).

Further, it may also be possible to prevent the adsorption of theamplification product to the three-dimensional porous member withoutperforming the separation with a moving part. An ultrafiltrationmembrane having a pore size of approximately 5 nm can be used as themembrane for preventing bead outflow 918 in place of ISOPORE having a0.8 μm pore size. In this case, the two-dimensional array chip and thethree-dimensional porous member remain in close contact with each otherduring all of the steps; however, the application of negative pressureis turned off at the time of the amplification reaction. Thus, the speedat which the amplification product passes through the ultrafiltrationmembrane is greatly reduced. It is thereby possible to prevent theadsorption of the amplification product to the three-dimensional porousmember.

Example 3

The present example describes the apparatus structure which combines afluorescence microscope for observation of the captured cell with thedevice structure of Example 1.

A structural view is shown in FIG. 10. The configuration of a device1001 is based on the basic configuration of Example 1. Note that theconfiguration of the device 1001 includes the three-dimensional porousmembers 319, 819, and 919 for high speed suction. Namely, the gravitydirection 217 is set in the direction opposite to the cell suctiondirection 216. The two-dimensional array chip 513 in the figure showsthe state during the sucking of the cell solution, and is in closecontact with the three-dimensional porous members 319, 819, and 919. 501is a captured cell.

Further, a diaphragm pump 1002 is used for the application of thenegative pressure. The cells suspended in the buffer in a tube 1003 forthe cell solution are introduced into the reaction chamber through thecell introduction port 511 by sucking with a syringe 1004. The excesscell solution and reagent are discharged in a tube 1006 for waste liquidthrough the sample discharge port 512 and a three-way stopcock 1005. Thediaphragm pump 1002 is operated in a state in which the cell solution isintroduced into the reaction chamber to capture the cells. To observethe state of at this time with the fluorescence microscope, an objectivelens 1007, a laser light source 1008 having an excitation wavelengthsuitable for a fluorophore which stained the cells, a dichroic mirror1009 for separating the excitation light and fluorescence from thecells, and a CCD camera 1010 as an imaging camera are provided as shownin FIG. 10. 1011 is an XYZ stage for focus adjustment and changing theimage region. When observing cells with a fluorescence microscope, it isnecessary to subject the cells to fluorescent staining. Here, a stainingmethod for staining the cell membrane is used. To acquire thefluorescence image via the microscope observation window 1012 on thedevice 1001, the material of the device including the window usesAcrylic (PMMA). As a matter of course, it is also possible to use othertransparent resin materials (polycarbonate, cycloolefin and the like).

All publications, patents, and patent applications cited herein areherein incorporated by reference in their entirety.

DESCRIPTION OF REFERENCE CHARACTERS

-   101, 201, 301: Cell-   102: Porous membrane-   103, 203, 303: Cell capture part-   104, 204, 304: Upper region-   105: Lower region-   106: Inlet-   107, 207, 307: Upper outlet-   108: Lower outlet-   109: Cell-   210, 310: Nucleic acid capture part-   211, 311: Cell introduction port-   212, 312: Sample collection port-   213, 313: Two-dimensional array chip-   214, 314: Lower region for holding cell solution prior to suction-   215, 315: Upper region-   216: Cell suction (force) direction-   217: Gravity direction-   318: Membrane for preventing bead outflow (hydrophilic porous    membrane)-   319: Solution holding part (three-dimensional porous member)-   320: Pressure adjustment member-   321: Intake port-   322: Airflow-   323: Storage tank-   333: Syringe-   500, 700, 800, 900: Device-   501, 701, 801, 901: Cell-   503, 703, 803, 903: Cell capture part-   507, 707: Lower outlet-   510, 810, 910: Nucleic acid capture part-   511, 711, 811, 911: Cell introduction port (upper inlet)-   512, 712, 812, 912: Sample collection port (upper outlet)-   513, 713, 813, 913: Two-dimensional array chip-   518, 718, 818: Membrane for preventing bead outflow-   524, 724, 824, 924: Reaction chamber-   525, 725, 825, 925: Common flow channel-   526, 726, 826, 926: Upper flow channel-   527, 727, 827, 927: Separate inlet-   528, 728, 828, 928: Region between reaction chambers-   601: First DNA probe-   602: Cell recognition sequences-   604: Universal primer-   606: Nucleic acid (mRNA)-   607: 1st strand-   608: Second DNA probe-   609: Universal sequence for amplification (Reverse)-   610: Chip identification tag-   611: Gene-specific sequence-   612: 2nd strand-   613: Universal primer-   614: Universal sequence for amplification (Forward)-   615. Amplification product-   729: Upper electrode-   730: Lower electrode-   731: Gold microparticle nucleic acid capture part-   732: High frequency power supply-   819, 919: Solution holding part (three-dimensional porous member)-   823: Storage tank-   833: Syringe-   834,934: Air valve-   835,935: Intake port-   836,936: Pressure adjusting filter-   837,937: Lower flow channel-   838,938: Lower outlet-   939: Air bag-   940: Syringe-   1001: Device (housing)-   1002: Diaphragm pump-   1003: Tube for cell solution-   1004: Syringe-   1005: Three-way stopcock-   1006: Tube for waste liquid-   1007: Objective lens-   1008: Laser light source-   1009: Dichroic mirror-   1010: CCD camera-   1011: XYZ stage-   1012: Microscope observation window

The invention claimed is:
 1. A cell analysis method using a cell analysis device which includes: a solution introduction channel, a two-dimensional array chip disposed above the solution introduction channel and having a plurality of cell capture parts and a plurality of nucleic acid capture parts, the cell capture parts being provided in communication with the solution introduction channel and each being capable of capturing a single cell, and each of the nucleic acid capture parts being provided in communication with a corresponding cell capture part of the plurality of cell capture parts and having a porous material or beads on which a nucleic acid probe configured to capture nucleic acid extracted from the single cell having been captured by the corresponding cell capture part, and a discharge channel disposed above the two-dimensional array chip in communication with the plurality of cell capture parts and having a three-dimensional porous member, the cell analysis method comprising: introducing a solution containing cells to the solution introduction channel; applying a negative pressure to the discharge channel to suck the solution from the solution introduction channel and adsorb the cells on the cell capture parts, the negative pressure bringing the two-dimensional array chip into contact with the three-dimensional porous member of the discharge channel; maintaining the negative pressure while extracting and capturing nucleic acid with the nucleic acid probe in the nucleic acid capture parts; introducing a reagent for nucleic acid amplification to the solution introduction channel; applying a negative pressure to the discharge channel to suck the reagent from the solution introduction channel and introduce the reagent to the nucleic acid capture parts, the negative pressure bringing the two-dimensional array chip into contact with the three-dimensional porous member of the discharge channel; separating the two-dimensional array chip from the three-dimensional porous member; and after the two-dimensional array chip is separated from the three-dimensional porous member, starting an amplification reaction with the reagent and the captured nucleic acid.
 2. The cell analysis method according to claim 1, wherein the two-dimensional array chip is separated from the three-dimensional porous member by stopping application of the negative pressure to the discharge channel.
 3. The cell analysis method according to claim 1, wherein the two-dimensional array chip includes an ultrafiltration membrane or a gel membrane is disposed above the nucleic acid capture parts and has a pore size smaller than a molecular size of a product of the amplification reaction.
 4. The cell analysis method according to claim 1, wherein the two-dimensional array chip is separated from the three-dimensional porous member by introducing a separation solvent or air between the two-dimensional array chip and the three-dimensional porous member.
 5. The cell analysis method according to claim 1, wherein the two-dimensional array chip is separated from the three-dimensional porous member by an actuator connected to the three-dimensional porous member.
 6. The cell analysis method according to claim 1, further comprising: collecting a product of the amplification reaction from the introduction channel by suction.
 7. The cell analysis method according to claim 1, wherein the applied negative pressure sucks the solution upwards counter to gravity from the solution introduction channel into the discharge channel.
 8. The cell analysis method according to claim 1, wherein the applied negative pressure to sucks the reagent upwards counter to gravity from the solution introduction channel into the discharge channel. 